Methods of treating or preventing pruritis by blocking natriuretic polypeptide b

ABSTRACT

Disclosed is a method of treating, reducing, or preventing pruritis in a mammal, the method comprising administering at least one natriuretic polypeptide b (Nppb) blocking agent to a mammal in an amount effective to treat or prevent pruritis in the mammal. An in vitro method of identifying a compound that inhibits Nppb activity is also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. application Ser. No.15/039,982, filed May 27, 2016, which is the U.S. national stage ofPCT/US2014/068541, filed Dec. 4, 2014, which claims the benefit of U.S.Provisional Patent Application No. 61/912,334, filed Dec. 5, 2013, eachof which is incorporated by reference herein in its entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: one 49,152 Byte ASCII (Text) file named“739188_ST25.txt,” dated May 4, 2018.

BACKGROUND OF THE INVENTION

Itch (also known as pruritis) is a sensation that may be perceived as anunpleasant skin irritation and may drive an urge to scratch. Some itchis transient and is no more than moderately unpleasant. In some cases,however, itch can become chronic, significantly reducing quality oflife. Conditions such as, for example, psoriasis, atopic dermatitis,renal failure, liver cirrhosis and some cancers may cause persistentitch. Accordingly, there is a need for improved compositions and methodsfor treating pruritis.

BRIEF SUMMARY OF THE INVENTION

An embodiment of the invention provides a method of treating, reducing,or preventing pruritis in a mammal, the method comprising administeringat least one natriuretic polypeptide b (Nppb) blocking agent(antagonist) to the mammal in an amount effective to treat, reduce, orprevent pruritis in the mammal.

Another embodiment of the invention provides a method of treating,reducing, or preventing pruritis in a mammal, the method comprisingadministering at least one Nppb blocking agent to the mammal in anamount effective to treat, reduce, or prevent pruritis in the mammal,wherein the Nppb blocking agent is not a Nppb-saporin conjugate.

Still another embodiment of the invention provides an in vitro method ofidentifying a compound that inhibits Nppb activity, the methodcomprising: (a) transducing one or more tester cells and one or morecontrol cells with at least one nucleotide sequence encoding aconstitutive reporter gene and natriuretic polypeptide receptor A (Npra)comprising a nucleotide cyclase domain, wherein the nucleotide cyclasedomain converts adenosine triphosphate (ATP) to cyclic adenosinemonophosphate (cAMP) and the constitutive reporter gene is under thetranscriptional control of cyclic adenosine monophosphate (cAMP); (b)contacting the tester cells of (a) with a test agent and Nppb; (c)contacting the control cells of (a) with Nppb; (d) incubating the cells;and (e) measuring the amount of reporter gene expression in the cells of(b) and (c), wherein a reduction in reporter gene expression in thecells of (b) as compared to the reporter gene expression in the cells of(c) is indicative of a compound that inhibits Nppb activity.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1A is a graph showing the numbers of bouts of scratching forwild-type mice (unshaded bars) and TRPV1-DTA mice (shaded bars) afterinjection with one of itch agents histamine, chloroguine, ET-1, 5HT,PAR2, or 48/80. Data are mean±standard error of the mean (s.e.m) (n≥7animals) normalized to wild-type litter controls. Behavioral responsesin TRPV1-DTA mice were all statistically different from responses ofwild-type control animals (Student's t-test, P<0.001).

FIG. 1B is a schematic representation showing the disruption of the Nppbgene by insertion of a splice acceptor-lacZ cassette into the secondexon which was used to generate Nppb^(−/−) mice.

FIG. 1C is a graph showing the normalized response (%) of Nppb^(−/−)mice to thermal, nocieptive, touch, and proprioceptive stimulation instandard assays. Data are mean s.e.m (n≥7 animals) normalized towild-type litter controls.

FIG. 1D is a graph showing the numbers of bouts of scratching forwild-type and Nppb^(−/−) mice after injection with one of itch agentshistamine, chloroguine, ET-1, 5HT, PAR2, or 48/80. For each item on they-axis, the left bar indicates wild-type, and the right bar indicatesNppb^(−/−) mice. Data are mean±s.e.m (n>7 animals) normalized towild-type litter controls. Behavioral responses in Nppb knockout micewere statistically different from responses of wild-type control animals(Student's t-test, P<0.001).

FIG. 1E is a graph showing numbers of bouts of scratching in wild-typeor Nppb^(−/−) mice after injection with Nppb or gastrin releasingpeptide (GRP). Data represent mean values±s.e.m. (n≥5 animals). “ns”=notsignificant (Student's T-test).

FIGS. 2A and 2B are graphs showing the number of Npra-expressing neurons(A) and GRPR-expressing neurons (B) per section in untreated mice ormice treated with Nppb-saporin or GRP-saporin. Data are mean±s.e.m.Significant differences between treatment groups were determined usingStudent's t-test with * indicating P<0.0001.

FIG. 2C is a graph showing the normalized response (%) of Nppb-saporintreated mice to thermal, nocieptive, touch, and proprioceptivestimulation in standard assays. Data represent means normalized againstuntreated controls±s.e.m. (n≥5 animals).

FIGS. 2D and 2E are graphs showing numbers of bouts of scratching afterhistamine (D) or Nppb (E) injection in untreated and Nppb-saporintreated mice. Data represent means normalized against untreatedcontrols±s.e.m. (n≥5 animals). Significant differences between genotypeswere determined using Student's t-test with * indicating P<0.01.

FIGS. 3A and 3B are graphs showing fold change of expression of GRP andNppb in the dorsal root ganglia (DRG) (A) and spinal cord (B). Datarepresent mean±s.e.m for triplicate cDNA preparations each analyzed intwo separate polymerase chain reaction (PCR) reactions. Transcriptlevels in DRG for GRP and Nppb were statistically different from eachother (Student's t-test, P<0.001) (FIG. 3A). Transcript levels in dorsalhorn (DH) of spinal cord for GRP and Tac1 were both statisticallydifferent from the amount of Nppb (Student's t-test, P<0.001) (FIG. 3B).

FIG. 3C is a graph showing the number of GRP neurons per section inuntreated mice and those treated with Nppb-saporin or GRP-saporin. Datarepresent mean±s.e.m. (n>4 animals). Significant differences betweengroups were determined using Student's t-test with * indicating P<0.001.

FIG. 3D is a graph showing numbers of bouts of scratching in untreatedmice and mice treated with Nppb-saporin after injection with GRP. Dataare mean±s.e.m. (n≥6 animals) and * indicates P<0.001 (Student'st-test).

FIG. 3E is a graph showing numbers of bouts of scratching in untreatedmice and those treated with GRP antagonist or GRP-saporin afterinjection with Nppb. Data are mean±s.e.m. (n≥6 animals) and * indicatesP<0.001 (Student's t-test).

FIG. 3F is a schematic model of the first three stages of thepruriceptive circuit with the neuropeptide used at each stage indicated.

FIGS. 4A and 4B are graphs showing numbers of bouts of scratching afterhistamine (A) or GRP (B) injection in untreated mice or mice treatedwith GRP antagonist or GRP-saporin. Data represent mean±s.e.m. (n≥5animals). Significant differences between treatment groups weredetermined using Student's t-test with * indicating P<0.001.

FIG. 5 is a graph showing numbers of bouts of scratches in mice thatwere treated with histamine alone, a combination of histamine and GRPantagonist, or a combination of histamine and anantin. Behavioralresponses to histamine were statistically different from responses tohistamine in the presence of either GRP-antagonist or Anantin (Student'st-test, P<0.01; n=5 mice).

FIG. 6A is a graph showing the numbers of bouts of scratches in micethat were intravenously (i.v.) administered PBS or various doses of Nppband untreated (shaded circles) or treated with GRP-antagonist (opencircles) or Nppb-saporin (squares). Behavioral responses elicited byi.v. injection of Nppb were statistically different from those ofanimals pretreated either with GRP-antagonist or Nppb-saporin (Student'st-test, P<0.05; n≥5 mice).

FIG. 6B is a graph showing the numbers of bouts of scratches inNppb-deficient or wild-type mice that were i.v. administered Nppb,natriuretic peptide A (Nppa), or natriuretic peptide C (Nppc).Behavioral responses elicited by i.v. injection of Nppb werestatistically different from those induced by administration of Nppa andNppc (Student's t-test, P<0.01; n=4-5 mice). The difference in responsesto i.v. injected Nppb between Nppb knockout mice and wild-type controlswas not statistically significant (Student's t-test, P>0.05; n=4-5mice).

FIG. 7 is a graph showing the numbers of bouts of scratches inwild-type, Nppb knockout, or Nppb-saporin-treated mice treated withIL-31. Behavioral responses of wild-type mice to IL-31 mice werestatistically different from those in wild-type control animalspretreated with Nppb-saporin and to responses observed for Nppb knockoutmice (Student's t-test, P<0.005; n=5 mice).

DETAILED DESCRIPTION OF THE INVENTION

Natriuretic polypeptide b (Nppb) (also known as brain natriureticpeptide or BNP) is a member of the natriuretic peptide family andencodes a secreted protein which functions as a cardiac hormone. Thebiological actions of Nppb may include natriuresis, diuresis,vasorelaxation, inhibition of renin and aldosterone secretion, andcardiovascular homeostasis. The Nppb receptor is natriuretic polypeptidereceptor A (Npra) (also known as atrial natriuretic peptide receptor A(NPR1)), a membrane-bound guanylate cyclase.

It has been discovered by the inventors of this invention that Nppb isrequired for pruriception and that administering an Nppb blocking agenttreats pruritis. Accordingly, an embodiment of the invention provides amethod of treating, reducing, or preventing pruritis in a mammal, themethod comprising administering at least one Nppb blocking agent to themammal in an amount effective to treat, reduce, or prevent pruritis inthe mammal.

The pruritis may be transient or chronic. Preferably, the pruritis ischronic.

The pruritis may be caused by or associated with any condition or anytreatment of a condition. In an embodiment of the invention, thepruritis may be caused by or associated with a skin condition. Examplesof skin conditions may include, but are not limited to, skin infectionsfrom Trichomonas or a fungus, psoriasis, and atopic dermatitis (alsoknown as eczema). In an embodiment of the invention, the pruritis may becaused by or associated with a systemic condition or treatment of asystemic condition. Examples of systemic conditions may include, but arenot limited to, renal failure, liver damage, liver disease (e.g.,cirrhosis), acquired immune deficiency syndrome (AIDS), polycythemiavera, diabetes, hyperthyroidism, and cancer (e.g., Hodgkin's lymphoma,non-Hodgkin's lymphoma, and Kaposi's sarcoma). Examples of treatments ofsystemic conditions include, but are not limited to, kidney dialysis andchemotherapy with agents such as, for example, doxorubicin,daunorubicin, cytarabine, paclitaxel, and cisplatin. Thesechemotherapeutic agents, which are used to treat a variety of cancers,may cause a skin reaction and may be associated with pruritus. Theincidence of non-cancer causes of pruritis may depend on the conditionand type of treatment.

The pruritis may be induced by a pruritogen. In an embodiment of theinvention, the pruritis is induced by a pruritogen selected from thegroup consisting of histamine, chloroquine, endothelin (ET-1), 2-methylserotonin (5HT), SLIGRL-NH2 (PAR2), and compound 48/80 (48/80).

The pruritis may be induced by a cytokine. In an embodiment of theinvention, the pruritis is induced or mediated by interleukin (IL)-31.IL-31 is associated with chronic itch in some types of skin disorderssuch as, for example, atopic dermatitis.

The Nppb blocking agent can be any agent that inhibits or reduces thebiological activity of Nppb. The biological activity of Nppb may beinhibited in any manner, e.g., by inhibiting the production (e.g.,expression) of any one or more of Nppb mRNA, Nppb protein, Npra mRNA,and Npra protein; by inhibiting the binding of Nppb to Npra, and/or byinhibiting Nppb signaling, as compared to that which is observed in theabsence of the Nppb blocking agent. The biological activity may beinhibited to any degree that realizes a beneficial therapeutic effect.For example, in some embodiments, the biological activity may becompletely inhibited (i.e., prevented), while in other embodiments, thebiological activity may be partially inhibited (i.e., reduced). As usedherein, unless stated otherwise, the terms “Nppb” and “Npra” refer toNppb and Npra, respectively, in any form (e.g., mRNA or protein) andfrom any species (e.g., human or mouse).

In an embodiment of the invention, the Nppb blocking agent is an agentthat inhibits Nppb signaling. Nppb signaling can be inhibited in anymanner. For example, the Nppb blocking agent may inhibit the activationof any one or more of various downstream targets of Nppb signaling(e.g., gastrin releasing peptide (GRP)). For example, the Nppb blockingagent may be an agent that binds to the Nppb protein, thereby reducingor preventing Nppb signaling and inhibiting its function. By way ofillustration, the agent that inhibits Nppb signaling can be any of theantibodies or antibody fragments, antisense nucleic acids, or chemicalinhibitors (e.g., small molecule or peptide (or polypeptide) inhibitor)described herein.

In an embodiment, the Nppb blocking agent is an agent that inhibits thebinding of Nppb to the Nppb receptor (Npra). In this regard, the Nppbblocking agent may be an agent that binds to the Nppb protein or theNpra protein, thereby reducing or preventing the binding of the Nppbprotein to Npra and inhibiting its function, as well as agents thatcompete with the Nppb protein for the native Nppb binding site of theNppb receptor (Npra). By way of illustration, the agent that inhibitsthe binding of Nppb to Npra can be any of the antibodies or antibodyfragments, antisense nucleic acids, or chemical inhibitors (e.g., smallmolecule or peptide inhibitor) described herein.

In an embodiment of the invention, the Nppb blocking agent is anantibody or antibody fragment that specifically binds to Nppb or Npra.Anti-Nppb and anti-Npra antibodies and antibody fragments can bemonoclonal or polyclonal. Anti-Nppb and anti-Npra antibodies andantibody fragments can be prepared using the Nppb and Npra proteinsdisclosed herein and routine techniques. Examples of such antibodies orantibody fragments include those specific to the native Nppb bindingsite of the Nppb receptor or a functional domain of Nppb (e.g., the Nprabinding portion of Nppb).

Chemical inhibitors of Nppb include small molecules and peptides orpolypeptides that inhibit Nppb signaling, bind the Nppb or Npra proteinor functional fragment thereof, or compete with the Nppb protein orfunctional fragment thereof for its native binding site of the Npra.Suitable inhibitors can include, for example, chemical compounds or anon-active fragment or mutant of an Nppb protein. In this regard, in anembodiment of the invention, the Nppb blocking agent is a mutated Nppb.The mutation may include any insertions, deletions, and/or substitutionsof one or more amino acids in any position of the Nppb protein thateffectively inhibits Nppb biological activity (e.g., Nppb signalingand/or binding of Nppb to Npra). For example, one or more native aminoacid residues in the Nppb protein (for example, those involved inreceptor binding) may be substituted with amino acid residues containingnon-natural side chains and/or D-amino acid residues. For example, thechemical inhibitor can bind to the Npra and/or inhibit Nppb signaling.In this regard, the Nppb blocking agent may be a chemical inhibitor. Ina preferred embodiment, the Nppb blocking agent inhibits the activationof gastrin releasing peptide (GRP). Examples of chemical inhibitorsinclude, but are not limited to, anantin, [Asu7,23′]b-ANP-(7-28)],HS-142-1, and a combination of any two or more thereof. In a preferredembodiment, the Nppb blocking agent is anantin.

Chemical inhibitors of Nppb can be identified using routine techniques.For example, chemical inhibitors can be tested in binding assays toidentify molecules and peptides (or polypeptides) that bind to Nppb orNpra with sufficient affinity to inhibit Nppb biological activity (e.g.,binding of Nppb to Npra, and/or Nppb signaling). Also, competitionassays can be performed to identify small-molecules and peptides (orpolypeptides) that inhibit the activation of downstream targets of Nppbsignaling or compete with Nppb or functional fragment thereof forbinding to its native binding site of Npra. Such techniques could beused in conjunction with mutagenesis of the Nppb protein or functionalfragment thereof itself, and/or with high-throughput screens of knownchemical inhibitors. For example, one or more native amino acid residuesin the Nppb protein may be randomly substituted with amino acid residuescontaining non-natural side chains and/or D-amino acids and the mutatedproteins can be tested in binding assays to identify mutated proteinsthat inhibit Nppb biological activity (e.g., binding of Nppb to Npra,and/or Nppb signaling).

The functional fragment of the Nppb or Npra protein can comprise anycontiguous part of the Nppb or Npra protein that retains a relevantbiological activity of the Nppb or Npra protein, e.g., binds to Npra orNppb and/or participates in Nppb signaling. Any given fragment of anNppb or Npra protein can be tested for such biological activity usingmethods known in the art. For example, the functional fragment cancomprise, consist essentially of, or consist of the Npra binding portionof the Nppb protein or the Nppb binding portion of the Npra protein. Inreference to the parent Nppb or Npra protein, the functional fragmentpreferably comprises, for instance, about 10% or more, about 25% ormore, about 30% or more, about 50% or more, about 60% or more, about 80%or more, about 90% or more, or even about 95% or more of the parent Nppbprotein.

In an embodiment of the invention, the Nppb blocking agent is anysuitable agent that inhibits the production (e.g., expression) of anyone or more of Nppb mRNA, Nppb protein, Npra mRNA, and Npra protein. TheNppb blocking agent can be a nucleic acid at least about 10 nucleotidesin length that specifically binds to and is complementary to a targetnucleic acid encoding any one or more of Nppb mRNA, Nppb protein, NpramRNA, and Npra protein or a complement thereof. The Nppb blocking agentmay be introduced into a host cell, wherein the cell is capable ofexpressing any one or more of Nppb mRNA, Nppb protein, Npra mRNA, andNpra protein, in an effective amount for a time and under conditionssufficient to interfere with production (e.g., expression) of any one ormore of Nppb mRNA, Nppb protein, Npra mRNA, and Npra protein,respectively. In some embodiments, RNA interference (RNAi) is employed.In this regard, the Nppb blocking agent may comprise an RNAi agent. Inan embodiment, the RNAi agent may comprise a small interfering RNA(siRNA), a short hairpin miRNA (shMIR), a microRNA (miRNA), or anantisense nucleic acid. The RNAi agent, e.g., siRNA, shRNA, miRNA,and/or antisense nucleic acid can comprise overhangs. That is, not allnucleotides need bind to the target sequence. RNA interference nucleicacids employed can be at least about 19, at least about 40, at leastabout 60, at least about 80, at least about 100, at least about 120, atleast about 140, at least about 160, at least about 180, at least about200, at least about 220, at least about 240, from about 19 to about 250,from about 40 to about 240, from about 60 to about 220, from about 80 toabout 200, from about 60 to about 180, from about 80 to about 160,and/or from about 100 to about 140 nucleotides in length.

The RNAi agent, e.g., siRNA or shRNA, can be encoded by a nucleotidesequence included in a cassette, e.g., a larger nucleic acid constructsuch as an appropriate vector. Examples of such vectors includelentiviral and adenoviral vectors, as well as other vectors describedherein with respect to other aspects of the invention. An example of asuitable vector is described in Aagaard et al. Mol. Ther., 15(5): 938-45(2007). When present as part of a larger nucleic acid construct, theresulting nucleic acid can be longer than the comprised RNAi nucleicacid, e.g., greater than about 70 nucleotides in length. In someembodiments, the RNAi agent employed cleaves the target mRNA. In otherembodiments, the RNAi agent employed does not cleave the target mRNA.

Any type of suitable siRNA, miRNA, and/or antisense nucleic acid can beemployed. In an embodiment, the antisense nucleic acid comprises anucleotide sequence complementary to at least about 8, at least about15, at least about 19, or from about 19 to about 22 nucleotides of anucleic acid encoding any one or more of Nppb mRNA, Nppb protein, NpramRNA, and Npra protein or a complement thereof. In an embodiment, thesiRNA may comprise, e.g., trans-acting siRNAs (tasiRNAs) and/orrepeat-associated siRNAs (rasiRNAs). In another embodiment, the miRNAmay comprise, e.g., a short hairpin miRNA (shMIR).

In an embodiment of the invention, the Nppb blocking agent may inhibitor downregulate to some degree the production of the protein encoded byan Npra or Nppb gene, e.g., at the DNA, RNA, or other level ofregulation. In this regard, a host cell comprising an Nppb blockingagent expresses none of any one or more of Nppb mRNA, Nppb protein, NpramRNA, and Npra protein or lower levels of any one or more of Nppb mRNA,Nppb protein, Npra mRNA, and Npra protein as compared to a host cellthat lacks an Nppb blocking agent. In accordance with an embodiment ofthe invention, the Nppb blocking agent, such as an RNAi agent, such as ashMIR, can target a nucleotide sequence of an Nppb or Npra gene or mRNAencoded by the same.

In an embodiment, the Nppb sequence is a human Nppb sequence. Forexample, human Nppb is assigned Gene NCBI Entrez Gene ID No. 4879, and aMendelian Inheritance in Man (MIM) No. 600295. The human Nppb gene isfound on chromosome 1 at 1p36.2. A transcript includes mRNA GenBankAccession No: NM_002521.2 (SEQ ID NO: 1), with corresponding proteinsequence GenBank Accession No: NP_002512.1 (SEQ ID NO: 2). Human genomicNppb sequences include GenBank Accession Nos: AC_000133.1, NC_018912.2,AB037521.1, ABBA01003061.1, AL021155.1, AMYH02000300.1, CH471130.1,EU326309.1, and M31776.1. Human Nppb mRNA sequences also include GenbankAccession Nos: AJ708502.1, BC025785.1, CR541976.1, CR542003.1, andM25296.1. Human Nppb amino acid sequences include Genbank Accession Nos:BAA90441.1, EAW71718.1, EAW71719.1, ACA05917.1, AAA35603.1, AAH25785.1,CAG46774.1, CAG46800.1, and AAA36355.1. Other human sequences, as wellas other Nppb species can be employed in accordance with the invention.

In an embodiment, the Npra sequence is a human Npra sequence. Forexample, human Npra is assigned Gene NCBI Entrez Gene ID No. 4881, and aMendelian Inheritance in Man (MIM) No. 108960. The human Npra gene isfound on chromosome 1 at 1q21-q22. Two transcriptional variants includemRNA GenBank Accession No: NM_000906.3 (SEQ ID NO: 3) and XM_005245218.1(SEQ ID NO: 5), with corresponding protein sequence GenBank AccessionNo: NP_000897.3 (SEQ ID NO: 4) and XP_005245275.1 (SEQ ID NO: 6),respectively. Human genomic Npra sequences include GenBank AccessionNos: AC_000133.1, NC_018912.2, AB010491.2, AB046472.1, ABBA01049444.1,AF190631.1, AL713889.19, AMYH02001904.1, AMYH02001905.1, CH471121.2, andEU326310.1. Human Npra mRNA sequences also include Genbank AccessionNos: AK025024.1, AK298090.1, AK300446.1, BC063304.1, S72628.1, andX15357.1. Human Npra amino acid sequences include Genbank Accession Nos:BAA31199.1, BAC53955.1, AAF01340.1, EAW53284.1, ACA05918.1, ACA05919.1,BAH12723.1, BAG62168.1, AAH63304.1, AAD14112.1, and CAA33417.1. Otherhuman sequences, as well as other Npra species can be employed inaccordance with the invention.

In another embodiment, the Nppb sequence is a mouse sequence. Forexample, mouse Nppb is assigned Gene NCBI Entrez Gene ID No. 18158. Themouse Nppb gene is found on chromosome 4 at 4 E2. A transcript includesmRNA Genbank Accession No.: NM_008726.4 (SEQ ID NO: 7), withcorresponding protein sequence NP_032752.1 (SEQ ID NO: 8). Mouse genomicNppb sequences include Genbank Accession Nos: NC_000070.6, NT_166299.2,AC_000026.1, AAHY01041891.1, AB039044.1, AB039045.1, AB039046.1,AB039047.1, AB039048.1, AB039049.1, AB039050.1, AB039051.1, AB039052.1,AL714013.9, CH466594.1, CU210867.6, D16497.1, D82049.1, and S58667.1.Mouse Nppb mRNA sequences also include Genbank Accession Nos:AK003128.1, BC061165.1, BU609640.1, and CK128345.1. Mouse Nppb aminoacid sequences include Genbank Accession Nos: BAB68568.1, BAB68569.1,BAB68570.1, BAB68571.1, BAB68572.1, BAB68573.1, BAB68574.1, BAB68575.1,BAB68576.1, EDL14790.1, BAA03948.1, BAA24159.1, AAB26344.2, BAB22588.1,and AAH61165.1. Other mouse sequences, as well as other Nppb species canbe employed in accordance with the invention.

In an embodiment, the Npra sequence is a mouse sequence. For example,mouse Npra is assigned Gene NCBI Entrez Gene ID No. 18160. The mouseNpra gene is found on chromosome 3 at 3 F1. A transcript includes mRNAGenbank Accession No.: NM_008727.5 (SEQ ID NO: 9), with correspondingprotein sequence NP_032753.5 (SEQ ID NO: 10). Mouse genomic Nprasequences include Genbank Accession Nos: NC_000069.6, AC_000025.1,AAHY01029027.1, AC145082.10, AJ307712.1, and CH466547.2. Mouse Npra mRNAsequences also include Genbank Accession Nos: AK135008.1, BC110659.1,BC139767.1, CJ065787.1, J05504.1, and L31932.1. Mouse Npra amino acidsequences include Genbank Accession Nos: CAC41350.1, EDL15140.1,BAE22383.1, AAI10660.1, AAA37670.1, and AAA66945.1. Other mousesequences, as well as other Npra species can be employed in accordancewith the invention. Human and mouse antisense nucleic acids arecommercially available (e.g., from OriGene Technologies, Inc.,Rockville, Md. or Sigma-Aldrich, St. Louis, Mo.) and can be preparedusing the nucleic acid sequences encoding the Nppb or Npra proteinsdisclosed herein and routine techniques.

In accordance with an embodiment of the invention, the Nppb blockingagent, such as an RNAi agent, such as a shMIR, can target a nucleotidesequence selected from the group consisting of the 5′ untranslatedregion (5′ UTR), the 3′ untranslated region (3′ UTR), and the codingsequence of Nppb or Npra, complements thereof, and any combinationthereof. Any suitable Nppb or Npra target sequence can be employed. Inan embodiment of the invention, the sequences of the Nppb blocking agentcan be designed against a human Nppb with the sequence of Accession No.NM_002521.2 (SEQ ID NO: 1). In an embodiment of the invention, thesequences of the Nppb blocking agent can be designed against human Nprawith either one of the sequences of Accession Nos: NM_000906.3 (SEQ IDNO: 3) or NM_005245218.1 (SEQ ID NO: 5), but also recognize the othersequence. In still another embodiment, the sequences of the Nppbblocking agent can be designed against a mouse Nppb with the sequence ofAccession No. NM_008726.4 (SEQ ID NO: 7) or a mouse Npra with thesequence of Accession No. NM_(——)008727.5 (SEQ ID NO: 9). RNAi agentscan be designed against any appropriate Nppb or Npra mRNA sequence.

In another embodiment, the Nppb blocking agent is an Nppb receptor/Fcfusion protein. The Nppb receptor/Fc fusion protein is a solublevariation of the native Npra which binds Nppb protein, thereby competingwith the native, cell surface Npra for binding to Nppb. Accordingly, theNppb receptor/Fe fusion protein may inhibit the binding of Nppb to thenative Npra. The Nppb receptor/Fe fusion protein may also inhibit theactivation of any one or more of various downstream targets of Nppbsignaling (e.g., GRP). The Nppb receptor/Fc fusion protein may be fromany mammal. In a preferred embodiment, the Nppb receptor/Fc fusionprotein is a mouse Nppb receptor/Fc fusion protein or a human Nppbreceptor/Fc fusion protein.

The Nppb blocking agent can be obtained by methods known in the art. Forexample, Nppb blocking agents that are peptides or polypeptides can beobtained by de novo synthesis as described in references, such as Chanet al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press,Oxford, United Kingdom, 2005; Peptide and Protein Drug Analysis, ed.Reid, R., Marcel Dekker, Inc., 2000; and U.S. Pat. No. 5,449,752. Also,Nppb blocking agents can be recombinantly produced using standardrecombinant methods. See, for instance, Sambrook et al., MolecularCloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, ColdSpring Harbor, N.Y. 2012. Further, the Nppb blocking agent can beisolated and/or purified from a natural source, e.g., a human. Methodsof isolation and purification are well-known in the art. In thisrespect, the Nppb blocking agents may be exogenous and can be synthetic,recombinant, or of natural origin.

The Nppb blocking agents that are peptides or polypeptides can beglycosylated, amidated, carboxylated, phosphorylated, esterified,N-acylated, cyclized via, e.g., a disulfide bridge, or converted into anacid addition salt and/or optionally dimerized or polymerized, orconjugated. Suitable pharmaceutically acceptable acid addition saltsinclude those derived from mineral acids, such as hydrochloric,hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids,and organic acids, such as tartaric, acetic, citric, malic, lactic,fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids,for example, p-toluenesulphonic acid.

Of course, the method of the invention can comprise administering two ormore Nppb blocking agents, any of which may be the same or differentfrom one another. Furthermore, the Nppb blocking agent can be providedas part of a larger polypeptide construct. For instance, the Nppbblocking agent can be provided as a fusion protein comprising an Nppbblocking agent along with other amino acid sequences or a nucleic acidencoding same. The Nppb blocking agent also can be provided as part of aconjugate or nucleic acid encoding same. Conjugates, as well as methodsof synthesizing conjugates in general, are known in the art (See, forinstance, Hudecz, F., Methods Mol. Biol. 298: 209-223 (2005) and Kirinet al., Inorg. Chem. 44(15): 5405-5415 (2005)).

In an embodiment of the invention, the Nppb blocking agent is notneurotoxic. In this regard, the Nppb blocking agent blocks thebiological activity of Nppb without ablating cells. In an embodiment,the Nppb blocking agent is not a Nppb-saporin conjugate. Accordingly, anembodiment of the invention provides a method of treating, reducing, orpreventing pruritis in a mammal, the method comprising administering atleast one natriuretic polypeptide b (Nppb) blocking agent to the mammalin an amount effective to treat, reduce, or prevent pruritis in themammal, wherein the Nppb blocking agent is not a Nppb-saporin conjugate.

The Nppb blocking agent can be administered to the mammal byadministering a nucleic acid encoding the Nppb blocking agent to themammal. “Nucleic acid” as used herein includes “polynucleotide,”“oligonucleotide,” and “nucleic acid molecule,” and generally means apolymer of DNA or RNA, which can be single-stranded or double-stranded,synthesized or obtained (e.g., isolated and/or purified) from naturalsources, which can contain natural, non-natural or altered nucleotides,and which can contain a natural, non-natural or altered internucleotidelinkage, such as a phosphoroamidate linkage or a phosphorothioatelinkage, instead of the phosphodiester found between the nucleotides ofan unmodified oligonucleotide.

Nucleic acids encoding the Nppb blocking agent (and degenerate nucleicacid sequences encoding the same amino acid sequences), can beconstructed based on chemical synthesis and/or enzymatic ligationreactions using procedures known in the art. See, for example, Sambrooket al., supra. For example, a nucleic acid can be chemically synthesizedusing naturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed upon hybridization(e.g., phosphorothioate derivatives and acridine substitutednucleotides).

The nucleic acids can be incorporated into a recombinant expressionvector. For purposes herein, the term “recombinant expression vector”means a genetically-modified oligonucleotide or polynucleotide constructthat permits the expression of an mRNA or polypeptide by a host cell,when the construct comprises a nucleotide sequence encoding the mRNA orpolypeptide, and the vector is contacted with the cell under conditionssufficient to have the mRNA or polypeptide expressed within the cell.The vectors are not naturally-occurring as a whole. However, parts ofthe vectors can be naturally-occurring. The recombinant expressionvectors can comprise any type of nucleotides, including, but not limitedto DNA and RNA, which can be single-stranded or double-stranded,synthesized or obtained in part from natural sources, and which cancontain natural, non-natural or altered nucleotides. The recombinantexpression vectors can comprise naturally-occurring ornon-naturally-occurring internucleotide linkages, or both types oflinkages. Preferably, the non-naturally occurring or altered nucleotidesor internucleotide linkages does not hinder the transcription orreplication of the vector.

The recombinant expression vector can be any suitable recombinantexpression vector, and can be used to transform or transfect anysuitable host. Suitable vectors include those designed for propagationand expansion or for expression or both, such as plasmids and viruses.The vector can be of the pUC series (Fermentas Life Sciences), thepBluescript series (Stratagene, LaJolla, Calif.), the pET series(Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala,Sweden), or the pEX series (Clontech, Palo Alto, Calif.). Bacteriophagevectors, such as λGT10, λGT11, kZapII (Stratagene), λEMBL4, and λNM1149,also can be used. Examples of plant expression vectors include pBI01,pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animalexpression vectors include pEUK-C1, pMAM and pMAMneo (Clontech).Preferably, the recombinant expression vector is a viral vector, e.g., aretroviral vector.

The recombinant expression vectors can be prepared using standardrecombinant DNA techniques described in, for example, Sambrook et al.,supra. Constructs of expression vectors, which are circular or linear,can be prepared to contain a replication system functional in aprokaryotic or eukaryotic host cell. Replication systems can be derived,e.g., from ColE1, 2μ plasmid, λ, SV40, bovine papilloma virus, and thelike.

Desirably, the recombinant expression vector comprises regulatorysequences, such as transcription and translation initiation andtermination codons, which are specific to the type of host (e.g.,bacterium, fungus, plant, or animal) into which the vector is to beintroduced, as appropriate and taking into consideration whether thevector is DNA- or RNA-based.

The recombinant expression vector can include one or more marker genes,which allow for selection of transformed or transfected hosts. Markergenes include biocide resistance, e.g., resistance to antibiotics, heavymetals, etc., complementation in an auxotrophic host to provideprototrophy, and the like. Suitable marker genes for the inventiveexpression vectors include, for instance, neomycin/G418 resistancegenes, hygromycin resistance genes, histidinol resistance genes,tetracycline resistance genes, and ampicillin resistance genes.

The recombinant expression vector can comprise a native or nonnativepromoter and/or stop codon operably linked to the nucleotide sequenceencoding the Nppb blocking agent, or to the nucleotide sequence which iscomplementary to the nucleotide sequence encoding the Nppb blockingagent. The selection of stop codons and promoters, e.g., strong, weak,inducible, tissue-specific and developmental-specific, is within theordinary skill of the artisan. Similarly, the combining of a nucleotidesequence with a stop codon and a promoter is also within the skill ofthe artisan. The promoter can be a non-viral promoter or a viralpromoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, anRSV promoter, and a promoter found in the long-terminal repeat of themurine stem cell virus.

The Nppb blocking agent and nucleic acids encoding them can be ofsynthetic or natural origin, and can be isolated or purified to anydegree. The terms “isolated” and “purified” as used herein means havingbeen increased in purity, wherein “purity” is a relative term, and notto be necessarily construed as absolute purity. For example, the puritycan be at least about 50%, can be greater than about 60%, about 70% orabout 80%, or can be about 100%.

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof pruritis in a mammal. Furthermore, the treatment or preventionprovided by the inventive method can include treatment or prevention ofone or more conditions or symptoms of the pruritis, e.g., chronicpruritis, being treated or prevented. Also, for purposes herein,“prevention” can encompass delaying the onset of the pruritis, or asymptom or condition thereof. With respect to the inventive methods, thepruritis can be any pruritis, including any of the types of pruritiscaused by or associated with any of the conditions or treatmentsdiscussed herein.

For purposes of the invention, the amount or dose of the Nppb blockingagent administered should be sufficient to effect the desired biologicalresponse, e.g., a therapeutic or prophylactic response, in the mammalover a reasonable time frame. The dose will be determined by theefficacy of the particular Nppb blocking agent and the condition of themammal (e.g., human), as well as the body weight of the mammal (e.g.,human) to be treated. The dose of the Nppb blocking agent also will bedetermined by the existence, nature and extent of any adverse sideeffects that might accompany the administration of a particular Nppbblocking agent. Typically, the attending physician will decide thedosage of the Nppb blocking agent with which to treat each individualpatient, taking into consideration a variety of factors, such as age,body weight, general health, diet, sex, Nppb blocking agent to beadministered, route of administration, and the severity of the conditionbeing treated.

The mammal referred to in the inventive methods can be any mammal. Asused herein, the term “mammal” refers to any mammal, including, but notlimited to, mammals of the order Rodentia, such as mice and hamsters,and mammals of the order Logomorpha, such as rabbits. It is preferredthat the mammals are from the order Carnivora, including Felines (cats)and Canines (dogs). It is more preferred that the mammals are from theorder Artiodactyla, including Bovines (cows) and Swines (pigs) or of theorder Perssodactyla, including Equines (horses). It is most preferredthat the mammals are of the order Primates, Ceboids, or Simoids(monkeys) or of the order Anthropoids (humans and apes). An especiallypreferred mammal is the human. The mammal can be non-diseased, a mammalafflicted with pruritis, or a mammal predisposed to pruritis.

Administering an Nppb blocking agent to the mammal in accordance withthe inventive methods may comprise administering a pharmaceuticalcomposition comprising the Nppb blocking agent and a pharmaceuticallyacceptable carrier. The carrier can be any of those conventionally usedand is limited only by chemico-physical considerations, such assolubility and lack of reactivity with the active compound(s), and bythe route of administration. The pharmaceutically acceptable carriersdescribed herein, for example, vehicles, excipients, and diluents, arewell-known to those skilled in the art and are readily available to thepublic. It is preferred that the pharmaceutically acceptable carrier beone which is chemically inert to the active agent(s) and one which hasno detrimental side effects or toxicity under the conditions of use. Thechoice of carrier will be determined in part by the particular compoundsused in the pharmaceutical composition, as well as by the particularmethod used to administer the Nppb blocking agent.

In an embodiment of the invention, administering the Nppb blocking agentto the mammal may comprise administering the Nppb blocking agent orally,intravenously, intramuscularly, subcutaneously, or intraperitoneally.The following formulations for oral, intravenous, intramuscular,subcutaneous, or intraperitoneal administration are exemplary and are inno way limiting. More than one route can be used to administer the Nppbblocking agent, and in certain instances, a particular route can providea more immediate and more effective response than another route.

Oral formulations may include any suitable carrier. For example,formulations suitable for oral administration may comprise suitablecarriers, such as lactose, sucrose, starch, talc magnesium stearate,crystalline cellulose, methyl cellulose, carboxymethyl cellulose,glycerin, sodium alginate or gum arabic among others.

Intravenous, intramuscular, subcutaneous, or intraperitonealformulations may include any suitable carrier. For example, formulationssuitable for intravenous, intramuscular, subcutaneous, orintraperitoneal administration may comprise sterile aqueous solutions ofthe Nppb blocking agent with solutions which are preferably isotonicwith the blood of the recipient. Such formulations may be prepared bydissolving the Nppb blocking agent in water containing physiologicallycompatible substances such as sodium chloride (e.g. 0.1-2.0M), glycine,and the like, and having a buffered pH compatible with physiologicalconditions to produce an aqueous solution, and rendering said solutionsterile.

An embodiment of the invention provides an in vitro method ofidentifying a compound that inhibits Nppb activity, the methodcomprising: (a) transducing one or more tester cells and one or morecontrol cells with at least one nucleotide sequence encoding aconstitutive reporter gene and Npra comprising a nucleotide cyclasedomain, wherein the nucleotide cyclase domain converts adenosinetriphosphate (ATP) to cyclic adenosine monophosphate (cAMP) and theconstitutive reporter gene is under the transcriptional control ofcyclic adenosine monophosphate (cAMP); (b) contacting the tester cellsof a) with a test agent and Nppb; (c) contacting the control cells of(a) with Nppb; (d) incubating the cells; and (e) measuring the amount ofreporter gene expression in the cells of (b) and (c), wherein areduction in reporter gene expression in the cells of (b) as compared tothe reporter gene expression in the cells of (c) is indicative of acompound that inhibits Nppb activity. In an embodiment, the method is ahigh-throughput method of identifying a compound that inhibits Nppbactivity.

The tester cells and control cells may be any suitable cell line. Themethod may comprise transducing the tester cells and control cells inany suitable manner known in the art with at least one nucleotidesequence encoding a constitutive reporter gene and Npra comprising anucleotide cyclase domain, wherein the nucleotide cyclase domainconverts ATP to cAMP and the constitutive reporter gene is under thetranscriptional control of cAMP.

The constitutive reporter gene may be any suitable constitutive reportergene known in the art. Examples of constitutive reporter genes include,but are not limited to, any of fluorescent protein (e.g., green (GFP),red, yellow, or cyan fluorescent protein, enhanced green, red, yellow,or cyan fluorescent protein), beta-lactamase, beta-galactosidase,luciferase (e.g., firefly luciferase (FLuc), Renilla (RLuc) luciferase,NANOLUC luciferase (NlucP) (Promega, Madison, Wis.), bacterialluciferase, Click-Beetle Luciferase Red (CBRluc), Click-BeetleLuciferase Green (CBG681uc and CBG991uc), Metridia pacifica Luciferase(MetLuc), Gaussia Luciferase (GLuc), Cypridina Luciferase, andGaussia-Dura Luciferase), chloramphenicol acetyltransferase (CAT),neomycin phosphotransferase, alkaline phosphatase, secreted alkalinephosphatase (SEAP), Chloramphenicol acetyltransferase (CAT), mCherry,tdTomato, TurboGFP, TurboRFP, dsRed, dsRed2, dsRed Express, AcGFP1,ZsGreen1, Red Firefly Luciferase, Enhanced Click-Beetle Luciferase(ELuc), Dinoflagellate Luciferase, Pyrophorus plagiophthalamusLuciferase (lucGR), Bacterial luciferase (Lux), pmeLUC, Phrixothrixhirtus Luciferase, Gaussia-Dura Luciferase, RenSP, Vargula hilgendorfiiLuciferase, Lucia Luciferase, Metridia longa Luciferase (MetLuc),HaloTag, SNAP-tag, CLIP-tag, ß-Glucuronidase, Aequorin, Secretedplacental alkaline phosphatase (SPAP), Gemini, TagBFP, mTagBFP2,Azurite, EBFP2, mKalama1, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean,SCFP3A, mTurquoise, mTurquoise2, Midoriishi-Cyan, TagCFP, mTFP1,Emerald, Superfolder GFP, Azami Green, TagGFP2, mUKG, mWasabi, Clover,Citrine, Venus, SYFP2, TagYFP, Kusabira-Orange, mKO, mKO2, mOrange,mOrange2, mRaspberry, mStrawberry, mTangerine, TagRFP, TagRFP-T, mApple,mRuby, mRuby2, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657,IFP1.4, iRFP, mKeima Red, LSS-mKate1, LSS-mKate2, PA-GFP, PAmCherry1,PATagRFP, Kaede (green), Kaede (red), KikGR1 (green), KikGR1 (red),PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), mEos3.2 (green), mEos3.2(red), PSmOrange, PSmOrange, Dronpa, TurboYFP, TurboFP602, TurboFP635,TurboFP650, hrGFP, hrGFP II, E2-Crimson, HcRed1, Dendra2, AmCyan1,ZsYellow1, mBanana, EBFP, Topaz, mECFP, CyPet, yPet, PhiYFP,DsRed-Monomer, Kusabira Orange, Kusabira Orange2, Jred, AsRed2,dKeima-Tandem, AQ143, mKikGR, and homologs and variants thereof.

The method may further comprise contacting the transduced tester cellswith one or more test agents and Nppb. In an embodiment, the testercells are contacted with a library of potential Nppb blocking agents(e.g., chemical inhibitors) and Nppb. The tester cells may be contactedwith one or more test agents and Nppb in any suitable manner. In anembodiment, the method comprises physically contacting the tester cellswith one or more test agents and Nppb. In an embodiment of theinvention, each of multiple subpopulations of tester cells is contactedwith a different test agent or combination of test agents in, forexample, multiwell plates.

The method may further comprise contacting the transduced control cellswith Nppb. The cells may be contacted with Nppb in any suitable manner.In an embodiment, the method comprises physically contacting the controlcells with Nppb.

The method may further comprise incubating the cells. The cells may beincubated under any suitable culture conditions known in the art for theparticular cells being used.

The method may further comprise measuring the amount of reporter geneexpression in the contacted, transduced tester cells and the contacted,transduced control cells. The amount of reporter gene expression may bemeasured in any suitable manner known in the art. A reduction inreporter gene expression in the tester cells as compared to the reportergene expression in the control cells is indicative of a compound thatinhibits Nppb activity.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLES Materials and Methods:

All experiments using animals followed NIH guidelines and were approvedby the National Institute of Dental and Craniofacial ACUC. The targetedJM8A3 ES-cell clone A04 with disruption of the Nppb gene was obtainedfrom Mouse Biology Program (MBP), UC Davis and was used to generatechimeric mice. Chimeras were crossed with C57BL/6 mice and heterozygousoffspring were mated to generate paired knockouts and controls.Transgenic (Tg) (gastrin releasing peptide (GRP)-enhanced greenfluorescent protein (EGFP)) animals (Gong et al., Nature, 425:917(2003)) were employed to localize expression of GRP. Male C57BL/6 (8-14weeks) mice were used for selective toxin ablation and GRP-antagonistitch experiments. Ablation of Npra and GRP-receptor-expressing spinalcord interneurons was accomplished by intrathecal (segment L3/4)injection of Nppb-saporin (5 μg in 10 μl; Advanced targeting Systems,San Diego, Calif.) and GRP-saporin (2.5 μg) respectively. Experimentswere initiated two weeks after toxin injection.

Itch inducing substances (see Table 1) were injected intradermally intothe shoulder of mice and numbers of scratching bouts assessed over 30minutes. Table 1 shows a list of pruritic agents and dose injectedsubcutaneously. Table 1 also indicates the receptors or mechanismsbelieved to be activated by the various compounds in the itch responsepathway.

Pruriceptive (itch) behavior was also elicited by lumbar 4-5 segmentintrathecal injection of Nppb (5 μg in 10 μl) or GRP (1 nM in 10 μl);just like responses to pruritogens, responses to peptides exhibiteddelayed onset of approximately (approx.) 5 minutes. Pretreatment withGRP antagonist deamino-Phe19,D-Ala24,D-Pro26-D-Phe27-GRP (Yegen et al.,Regulatory Peptides, 61:175 (1996)) (1 nM in 10 μl) was used to blockGRP-receptor.

TABLE 1 Pruritogen Dose (10 μl) Receptor Histamine 100 μg Histamine (H1)Chloroquine 100 μg MrgprA3 Endothelin (ET-1)  25 ng ETA 2-methylserotonin (5HT)  30 μg 5-HT SLIGRL-NH2 (PAR2) 100 μg MrgprC11 Compound48/80 (48/80) 100 μg Mast-cells

Thermal, mechanical, proprioceptive and pruriceptive behavioralresponses were assessed as described previously (Mishra et al., EMBO J.,30:582 (2011)). Thermal reactivity was determined using a hot plate (55°C.) or cold plate (−5° C.), with the time to the first lick or jumprecorded. Mechanical sensitivity was measured using a semi-automatedvon-Frey apparatus and Randall-Sellito device. Proprioceptive responseswere assessed using an accelerating rotarod. Statistical analysis usedPrism Graph; P>0.05 values were considered non-significant.

In situ hybridization (ISH) was performed at high stringency (washed 30minutes (min), 0.2×SSC, 70° C.) as described previously (Hoon et al.,Cell, 96:541 (1999), Adler et al., Cell, 100:693 (2000)). Detection ofMAS-related G-protein coupled receptor (GPR), member A(MrgprA)-receptors used a mix of full-length MAS-related GPR, member A3(MrgprA3) and MAS-related GPR, member A4 (MrgprA4) anti-sense probes.Nppb-expression outside the somatosensory system was examined and signalwas not detected in other sensory systems or the brain. As expected,Nppb was prominently expressed in the heart. Immunohistochemistry wasperformed with monoclonal antibody (mAb) anti-Nppb, rabbitanti-MAS-related GPR, member X1 (MrgprC11), and rabbit anti-Npra (fromLifeSpan Bioscience, Seattle, Wash.); chicken anti-GFP was from Abcam(Cambridge, England) and secondary antibodies were Jackson Immunolabs(West Grove, Pa.); tyramine FITC was used for HRP-signal amplificationto visualize Npra-immunostaining. Fluorescent images (1 μm opticalsections) were collected using confocal microscopy and were processedusing Adobe Photoshop.

Total RNA was extracted from dorsal root ganglia (DRG) and spinal cordusing an RNAeasy kit and converted into cDNA. Quantitative real-time PCRwas accomplished with commercially available TAQMAN primer sets. Equalamounts of cDNA were used in duplicate and amplification efficiencieswere validated and normalized against glyceraldehyde 3-phosphatedehydrogenase (GAPDH), fold increases were calculated using thecomparative threshold cycle method. Agilent whole genome arrays (mouseGE 44K V2) were screened with cRNA probes generated from DRG asrecommended by the manufacturer. Bioinformatic analysis was performedwith Gene-Spring software.

EXAMPLE 1

This example demonstrates that Nppb is co-expressed with itch relatedsignaling molecules TRPV1 and PLCβ3.

Mice were previously generated that had lost all transient receptorpotential cation channel, subfamily V, member 1 (TRPV1)-lineage neurons(Mishra et al., EMBO J., 30:582 (2011)). TRPV1-DTA mice exhibiteddramatically reduced scratch responses following intradermal injectionof pruritic agents histamine (100 μg in 10 μl); chloroquine (100 μg);endothelin 1 (1 μM); methyl-serotonin (30 μg); PAR2 agonist SLIGRL-NH2(100 μg) and compound 48/80 (100 μg). Itch-inducing substances wereinjected intradermally into the shoulder of mice and numbers ofscratching bouts were assessed over 30 minutes. As shown in FIG. 1A,these mice had major pruritic deficits as well as a complete loss ofthermosensory input (Mishra et al., EMBO J., 30:582 (2011)), in keepingwith previous reports using capsaicin-induced lesions (Imamachi et al.,PNAS, 106:11330 (2009), Cavanaugh et al., PNAS, 106:9075 (2009)).

To identify candidate molecules that might mediate itch signaling, adifferential microarray-based screen that identified many TRPV1-enrichedtranscripts (Table 2) was used. Table 2 shows the top 25 over-expressedgenes in wild-type versus (vs) TRPV1-DTA mice. In situ hybridization(ISH) of sections through the DRG showed the loss of Nppb-expression inTRPV1-DTA animals. Quantitation of Nppb expressing neurons revealed that7±0.6% of NeuN-positive C4 DRG neurons expressed the neuropeptide inwild type mice (n=6). Amongst the genes in Table 2, the natriureticpolypeptide b (Nppb) was prominently expressed in a small subset of DRGneurons, but was dramatically decreased in sensory ganglia fromTRPV1-DTA animals.

TABLE 2 Gene name Fold-change P-value Genbank acc # Apod 105 3.8e−5NM_007470 Trpv1 96 1.3e−4 NM_001001445 Myot 32 8.7e−6 NM_001033621Zcwpw2 32 7.5e−2 XM_001473321 Gfra3 32 2.1e−6 NM_010280 Tnnt3 28 1.9e−6NM_001163664 Osta 27 2.4e−5 NM_145932 Myl1 27 2.1e−4 NM_001113387Ceacam10 26 1.4e−4 NM_007675 Wfdc2 26 2.8e−5 NM_026323 Kcnf1 25 6.2e−4NM_201531 Trdn 25 4.3e−5 NM_001251987 Nppb 25 2.2e−6 NM_008726 Cacna2 259.7e−5 NM_001110843 Dgkk 25 2.1e−4 NM_177914 AW551984 25 1.0e−3NM_001199556 Syt16 21 4.4e−5 NM_172804 Cacna1i 21 6.7e−6 NM_001044308Gpr35 21 3.2e−4 NM_022320 9430021M05Rik 21 3.9e−5 NR_033569 Tnnc2 212.1e−6 NM_009394 Avpr1a 20 8.9e−4 NM_016847 Bex1 19 3.6e−6 NM_009052Trpa1 19 8.1e−7 NM_177781 P2rx3 18 1.6e−6 NM_145526

Double label ISH of DRG demonstrated that Nppb and TRPV1 wereco-expressed in the same sensory neurons. Only a subset of TRPV1expressing neurons contained Nppb. All Nppb-positive neurons alsoexpressed PLCβ3 but many PLCβ3 neurons were Nppb negative. Accordingly,double label ISH demonstrated that all Nppb-expressing neurons containedTRPV1 and PLCβ3, which are critically required for histamine-inducedscratching in mice (Imamachi et al., PNAS, 106:11330 (2009), Han et al.,Neuron, 52:691 (2006)).

Double-label ISH also showed that Nppb-positive neurons all expressedMrgprA receptors (including MrgprA3, the receptor for chloroquine), withmore than 70% of MrgprA expressing-neurons also containing theneuropeptide. Double-label immunostaining demonstrated complete overlapbetween expression of MrgprC11, the receptor for the pruritogenSLIGL-NH₂ and Nppb in somatosensory neurons. Accordingly, doublelabeling showed almost complete overlap between the expression of Nppband two Mas related G protein coupled receptors that have recently beenshown to detect specific pruritogens (Liu et al., Cell, 139:1373 (2009),Liu et al., Science Signaling, 4:ra45 (2011), Han et al., Nat.Neurosci., 16:174 (2012)).

Many itch-inducing agents also trigger a peripheral inflammatoryresponse. Therefore, double label in situ hybridization was used todetermine whether Nppb-expressing neurons in DRG also containneuropeptides that are known to be released in the periphery and causeneurogenic inflammation. Very few Nppb-positive cells co-expressedsubstance P (Sub P). However, about a quarter of Nppb-expressing neuronscontained CGRP and half of the Nppb-neurons expressed neuromedin B(NMB). Quantification of the Nppb positive neurons revealed that 2%co-labeled for Substance P, 24%, for CGRP and 50% for NMB (assessed fromtotal neuronal counts of 147, 185, and 196 respectively).

EXAMPLE 2

This example demonstrates the generation and characterization of Nppbmice.

Nppb^(−/−) animals were generated by inserting a splice acceptor-lacZcassette into the second Nppb exon, as shown in FIG. 1B. In situhybridization of sections through DRG revealed that Nppb expression waslost in Nppb^(−/−) animals, showing that these mutants displayed nodetectable expression of Nppb.

The mice were healthy and had normal numbers of nociceptive, touch, andproprioceptive neurons. The distribution and number of dorsal horninterneurons were unaffected by gene disruption. Sections through DRGand spinal cord from wild-type control and Nppb^(−/−) mice werehybridized with probes for Nppb and for genes expressing moleculesinvolved in pruriception, nociception, proprioception, mechanical, andthermal sensation (TRPV1, TRPA1, TRPM8, Mrgc11, tyrosine hydroxylase(TH), TrkB, TrkC, Npy2r, MrgD, Sst, Tac1, Npy, Gal, and Npra). Exceptfor Nppb, no significant changes between genotypes were observed innumbers of positive cells.

Nppb^(−/−) mice retained normal responses to thermal, nocieptive, touch,and proprioceptive stimulation when tested using standard assays (FIG.1C).

Intradermal injections were performed. The number of scratching boutsfor substances that directly activate pruriceptors was recorded. Thenumber of scratching bouts after injection with compound 48/80, whichcauses itch via an indirect route (McNeil et al., Neuroscience Bulletin,28:100 (2012)), was also recorded. All of these agents (Table 1)reliably triggered multiple bouts of scratching in control animals (FIG.1D), but Nppb^(−/−) mice were almost completely insensitive to the fullrange of pruritic substances tested (FIG. 1D).

How Nppb induces this stereotyped scratch response was investigated. Itwas hypothesized that because this peptide is prominently expressed insomatosensory neurons, the most plausible explanation for its role wouldbe if it acted as a specific itch-related neuromodulator (orneurotransmitter) in the spinal cord. Intrathecal injection of Nppb (5μg in 10 μl) into the lumbar 4-5 segment of control and Nppb miceinduced repeated bouts of scratching (FIG. 1E). Injection of GRP (1 nMin 10 μl) also triggered itch responses in both mutant and controlanimals (FIG. 1E). Indeed, intrathecal injection of Nppb inducedprofound scratching behavior in wild type animals (FIG. 1E),demonstrating that spinal-Nppb is sufficient to induce itch even withoutactivation of the peripheral neurons that express it. Intrathecalinjection of Nppb into Nppb^(−/−) mice also led to an equivalentphenotype (FIG. 1E). No significant differences in response betweengenotypes were found (Student's t-test). Loss of Nppb in sensoryafferents was thus responsible for the pruriceptive deficits seen inmutant mice. Without being bound to a particular theory or mechanism, itis believed that Nppb-expression delineates the subset of somatosensoryneurons that detect pruritic agents and that central release of Nppbfrom these neurons is necessary for the itch response.

EXAMPLE 3

This example demonstrates that selective ablation of Npra receptorneurons in the spinal cord impairs pruriception.

Because Nppb is responsible for transmitting the peripheral signals thattrigger pruritic responses, its receptor Npra (Misono et al., The FEBSJournal, 278:1818 (2011)) should be expressed at the site of afferentfiber synaptic connections in the spinal dorsal horn and mark thesecondary neurons in the itch response circuit. Therefore, expression ofNpra in the dorsal spinal cord was assessed using ISH. In normal mice, asignificant subset of interneurons in the outer layer express Npra,however after intrathecal administration of Nppb-saporin, few Npraneurons remained. In contrast, the number of GRP-receptor positive cellswere unaffected by Nppb-saporin. Therefore, it was found that Npra was,indeed, expressed in a limited subset of neurons (most likelyinterneurons, see below), primarily in the outer layer, i.e., lamina I,corresponding to the terminal field of TRPV1-expressing sensory neurons(Caterina et al., Science, 288:306 (2000)).

To examine whether the Npra neurons in the spinal cord function in theitch circuit and if they are selectively required for pruritogen-inducedscratching (rather than other somatosensory responses), a targeted-toxincell ablation strategy was used (Wiley et al., Advanced Drug DeliveryReviews, 55:1043 (2003)). An Nppb-saporin conjugate was injectedintrathecally into wild type mice to target their Npra-expressing cells,and the effectiveness, specificity and behavioral consequences ofadministering this toxin were assessed. ISH analysis of Nppb-saporintreated mice and untreated mice showed that intrathecal Nppb-saporintreatment had no effect on expression patterns of spinal cordneuropeptides Sst, Tac1, Npy, or Gal. Analysis of numbers of neuronsablated by Nppb-saporin (FIG. 2A) and GRP-saporin (FIG. 2B)administration showed that approximately 70% of Npra-positive cells wereeliminated by Nppb-saporin administration. By contrast, Nppb-saporininjection did not alter numbers of GRP-receptor positive neurons. ISHwas performed on tissue from at least 4 control and 4 treated mice.Serial sections from >10 sections per mouse were hybridized and numbersof cells counted. Accordingly, this targeted ablation of Npra receptorneurons was highly selective, with neither cells expressing theGRP-receptor nor other dorsal horn interneurons affected by Nppb-saporintreatment.

Toxin-injected mice displayed normal responses to thermal, touch, andpainful stimulation (FIG. 2C). However, a dramatic reduction inscratching evoked by histamine or Nppb (FIGS. 2D and 2E) was observed,indicating that these neurons are required for itch responses, but notfor other somatosensory pathways.

EXAMPLE 4

This example demonstrates that GRP acts downstream of Nppb in the rodentpruriceptive circuit.

The GRP-receptor has been shown to be a key element in the pruriticpathway (Sun et al., Nature, 448:700 (2007), Sun et al., Science,325:1531 (2009)) with the suggestion that GRP might be the primaryneurotransmitter for itch. However, this view has also been questioned(McNeil et al., Neuroscience Bulletin, 28:100 (2012), Fleming et al.,Mol. Pain, 8:52 (2012)). Quantitative PCR (qPCR) was used to quantitateexpression of GRP and Nppb relative to GAPDH in the DRG and spinal cord.GRP was robustly expressed in the spinal cord (at a level comparablewith Tac1) but was almost undetectable in the DRG (FIGS. 3A and 3B).Nppb was prominently expressed in DRG, but was not present in the spinalcord (FIGS. 3A and 3B). No more than trace quantities of GRP expressionwere detected in the DRG using a sensitive qPCR assay (FIG. 3A).

Similarly, somatosensory neurons from GRP-reporter mice Tg(GRP-EGFP)were negative for EGFP expression. ISH and immunohistochemistry (IHC) oftissue from Tg(GRP-EGFP) animals showed that GRP was expressed in apopulation of dorsal horn interneurons. In contrast, GRP was notexpressed by primary sensory neurons. As shown in FIG. 3C, a significantnumber of GRP-neurons was eliminated following Nppb-saporin (Nppb-sap)treatment. GRP-saporin (GRP-sap), which targets GRP-receptor neurons,had no effect on the number of GRP-interneurons. ISH revealed that Nppbwas expressed in DRG and was absent from the spinal cord. Therefore, itwas concluded that GRP cannot act at the level of pruriception, but mustfunction downstream of Nppb. Three complementary functional strategieswere applied to substantiate this hypothesis and dissect the itchresponse circuit.

First, it was demonstrated that GRP-induced scratching was unalteredeither by Nppb-knockout (FIG. 1E) or by the ablation of Npra-expressingneurons (FIG. 3D). Second, pharmacological inhibition of theGRP-receptor not only attenuated behavioral responses to the pruriticagent histamine or GRP injection (FIGS. 4A and 4B), but also inhibitedscratching after intrathecal administration of Nppb (FIG. 3E).Pretreatment with a GRP antagonist or ablation of GRP-receptorexpressing neurons with GRP-saporin attenuated scratching followingintradermal injection of histamine or intrathecal administration of GRP(FIGS. 4A and 4B). Knockout of the GRP-receptor had a much less severeeffect on behavioral-responses to histamine than pharmacologicalinhibition, suggesting that compensatory mechanisms may occur in GRP-Ranimals. These results show that killing the GRP-R expressing cells withGRP-saporin (Sun et al., Science, 325:1531 (2009)) more closelyresembles pharmacological inhibition of the receptor. Lastly, mice weretested with selective ablation of GRP-receptor-expressing neurons andagain found significantly reduced itch responses to Nppb (FIG. 3E). Asshown in FIG. 3E, scratching induced by lumbar injection of Nppb wasstrongly attenuated by pretreatment with a selective GRP antagonist orby the ablation of GRPR-expressing neurons with GRP-saporin.

These data place GRP downstream of Nppb in the itch response circuit.These data suggest that the secondary pruriceptors are targets for oneneuropeptide, Nppb, and, in turn, signal through a second peptide, GRP.Indeed, just as this model predicts, all Npra-expressing neurons in thedorsal horn contained GRP, and Nppb-saporin treatment significantlyreduced the number of GRP-expressing cells. ISH was used to analyze GRPexpression in the dorsal horn of normal and Nppb-saporin treated mice.Many GRP expressing interneurons were lost on ablation ofNpra-expressing cells. Double-label immunohistochemistry was used tolocalize interneurons expressing Npra and GRP-driven EGFP in sectionsthrough the dorsal horn of Tg(GRP-EGFP) mice.

These results molecularly characterized the first three stations of anitch response pathway in mice (FIG. 3F), demonstrated that Nppb marksthe primary sensory neurons and showed that this peptide is bothnecessary and sufficient for transmission of peripheral signals thatinduce stereotypic itch responses. Unlike previously characterizedreceptors (Liu et al., Cell, 139:1373 (2009), Liu et al., ScienceSignaling, 4:ra45 (2011)) and signaling molecules (Shim et al., J.Neurosci., 27:2331 (2007), Imamachi et al., PNAS, 106:11330 (2009), Hanet al., Neuron, 52:691 (2006)) that affect the detection of particularitch-inducing agents, Nppb is necessary for responses to a wide range ofpruritogens (i.e., compounds classed as inducing histamine andnon-histamine related itch, Table 1). These data also refine the roleGRP and GRP-receptor cells play in the itch response pathway by placingthem at later stages than had been hypothesized previously (Sun et al.,Nature, 448:700 (2007), Sun et al., Science, 325:1531 (2009)).

EXAMPLE 5

This example demonstrates that an Npra antagonist, anantin, attenuateshistamine-induced itch.

Wild-type mice (C57BL/6) were administered histamine alone, acombination of histamine and a GRP antagonist, or a combination ofhistamine and anantin. GRP is an antagonist to another receptor in theitch pathway and was used as a control. Bouts of scratching werecounted. The results are shown in FIG. 5. As shown in FIG. 5, anantinattenuated histamine-induced itch.

EXAMPLE 6

This example demonstrates that elevated blood Nppb can directly produceitch in a mouse model.

Elevated blood Nppb is found in kidney disalysis patients. Accordingly,the effect of elevated blood Nppb on itching in mice was evaluated.Wild-type mice (C57BL/6) were intravenously administered Nppb and wereuntreated or treated with GRP-antagonist or Nppb saporin. The bouts ofscratching were counted. The results are shown in FIG. 6A. As shown inFIG. 6A, the i.v. Nppb-induced itch was blocked with a GRP antagonist orby eliminating neurons that express the Npra receptor (Npra is the Nppbreceptor) with Nppb-saporin toxin conjugate.

Nppb deficient or wild-type (C57BL/6) mice were intravenously (i.v.)administered Nppb, Nppa, or Nppc, and the bouts of scratches weremeasured. The results are shown in FIG. 6B. As shown in FIG. 6B, inmouse, the elevated Nppb treatment was specific for the Nppb peptidebecause Nppa and Nppc did not induce significant itch. In addition, FIG.6B shows that in a mouse deficient for Nppb, i.v. injection of itchstill produced scratch responses, establishing that the Nppb acts on thecentral (CNS) pathway.

EXAMPLE 7

This example demonstrates that itching induced by the itch-inducingagent interleukin (IL)-31 also depends on Nppb signaling.

IL-31 is a cytokine that has been linked with skin conditions and isassociated with chronic itch in some types of skin disorders such as,for example, atopic dermatitis. To investigate the role of Nppb inIL-31-induced itching, wild-type (C57BL/6) (untreated withNppb-saporin), Nppb knockout, or Nppb-saporin treated C57BL/6 mice wereall treated with IL-31, and the numbers of bouts of scratches werecounted. The results are shown in FIG. 7. As shown in FIG. 7, eitherloss of Nppb or elimination of Npra-neurons leads to marked reduction inresponses to the itch agent IL-31. In addition, ISH studies revealedthat the receptor for IL-31 is found in the neurons that express Nppb,supporting the concept that Nppb signaling is required for a clinicallyrelevant type of itch.

EXAMPLE 8

This example demonstrates that administering Nppb to mice induces itch.

In addition to being a key component of the itch neural pathway, Nppb isalso produced by the heart and secreted into blood. Nppb controls bloodvolume and sodium, mainly by regulating sodium secretion by the kidney.In addition, Nppb is a standard biomarker used for diagnosis of heartfailure, where elevated Nppb can be measured for up to 48 hours (h)following infarction. Nppb may also be involved in regulating bloodvolume in response to stress. Very high blood Nppb concentrations arealso found in renal failure patients. Accordingly, it was investigatedwhether there is a causal link between the high circulatory Nppb anditch.

To mimic the high concentration of Nppb found in renal itch patients,mice were i.v. administered Nppb (via tail vein injection). Treated miceexhibited robust whole body scratching behavior. Approximately tenminutes after administration of Nppb, mice began to persistently scratchtheir flank, face, hind quarters and abdomens. This behavior slowlydiminished and almost completely ceased approximately 1 hour afterinjection. The induction of scratching was dose dependent, with a 1.2μg/kg dose producing saturating behavioral responses. In all subsequentexperiments, a dose of 1.2 μg/kg Nppb (abbreviated as “ivNppb”) wasused, and bouts of scratching were counted over a period of 1 hour.

It was previously demonstrated that pruriceptive specific spinal cordinterneurons express the Nppb-receptor, Npra, and are upstream of itchspecific neurons expressing the specific gastrin releasing peptidereceptor (GRPR). Npra neurons themselves express GRP and are thought torelease GRP which, in turn, activates GRPR-cells. Therefore, aGRP-antagonist was used to show that ivNppb-induced behavior(intrathecal administrated 5 minutes prior to iv Nppb) in factrepresented the behavioral correlate of itch-sensation. Notably, it wasalso found that injection of C-type natriuretic peptide (encoded by thegene Nppc), the selective agonist for the related natriurectic peptidereceptor Nprb, produced no observable reaction, showing that thisbehavior is very specific and indicating that this phenomenon isprobably dependent on the Npra. Supporting this hypothesis, ablation ofNpra-expressing neurons in the spinal cord with saporin conjugated Nppbtoxin eliminated Npra-expressing neurons and abolished ivNppbitch-behavior. Finally, as expected for a centrally acting mechanism,the genetic ablation of all peripheral itch sensory neurons, orelimination of Nppb in sensory neurons, did not affect ivNppb-inducedscratch.

EXAMPLE 9

This example demonstrates that high blood Nppb may causes chronic itchvia a neurogenic pathway.

To gain additional evidence that systemic Nppb activates a central itchpathway, the pattern of cellular activation of neurons in the dorsalroot ganglion (DRG) and superficial dorsal horn (sDH) was compared byanalyzing induction of immediate early gene expression. The skin wasstimulated by pruritogen and injections of noxious compounds and therobust induction of immediate early genes, Egr1 and cFos (in the DRG andsDH respectively) was evaluated. Intradermal injection of chloroquinewas utilized to excite MrgprA3 peripheral DRG neurons. As expected, ISHanalysis showed that administration of chloroquine triggereditch-behavior in mice and induced Egr1 expression in the cell-bodies ofa population of DRG neurons. The number of positive Egr-1-positive cellscorrelated well with the previously reported numbers ofMrgprA3-expressing receptor neurons in DRG. Also as expected, injectionof the noxious agent, formalin, induced Egr-1 expression in a far largernumber and morphologically heterogeneous class of sensory neurons,indicating that different populations of neurons were activated by thesedifferent sensory modalities. Likewise, chloroquine and formalin inducedcFos expression in different profiles of ipsilateral sDH neurons.Therefore, the initial and later cellular stages of the pruriceptiveneural pathway could be reliably monitored biochemically andanatomically and itch signaling could be distinguished from thatelicited by the noxious agent formalin.

It was reasoned that if ivNppb was directly activating centrally, theninduction of immediate early gene expression would be seen insDH-neurons and not DRG neurons. Indeed, ISH analysis showed that ivNppbcaused cFos induction in the sDH, but importantly did not induceexpression of Egr1 in the DRG. As expected from the generalizedscratching responses evoked by ivNppb, in both hemispheres of the spinalcord and along the entire length of the spinal cord, cFos-activatedneurons could be detected. Furthermore, also consistent with theitch-behavioral result, the profile of cFos-activated neurons in the sDHwas similar to that generated by epidermal injection of chloroquine.

Here, molecular, genetic and behavioral assays were used to examine thelink between systemic Nppb and itch sensation. Multiple lines ofevidence are presented showing that elevated blood Nppb can induce itchthrough central pathways. Without being bound to a particular theory ormechanism, it is believed that at certain concentrations, systemic Nppbmay gain access to the spinal cord and “inadvertently” stimulate itchsensory pathways. Nppb is a small molecule and may gain access to thespinal cord like a number of other small peptides such as opioids thathave been reported to gain access to the CNS from the periphery. Undernormal circumstances, the body controls blood volume and sodium bymaking fine adjustments to the level of Nppb. Without being bound to aparticular theory or mechanism, it is believed that during kidneyfailure, the body reacts to elevated blood sodium and blood volume bysecreting additional Nppb in an attempt to rectify imbalances in bloodsodium and volume. In this condition, a classical positive feedback maydevelop caused by Nppb-resistance of the kidney. Therefore, withoutbeing bound to a particular theory or mechanism, it is believed thathigh blood Nppb found in uremic itch patient is causal for chronic itchand explains why treatments targeting dermatological origin of itch arelargely ineffective. Furthermore, the results suggest that aNppb-receptor antagonist might be a therapeutic option for treatment ofuremic itch.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method of treating or reducing pruritis in a mammal in needthereof, the method comprising administering at least one natriureticpolypeptide b (Nppb) blocking agent to the mammal-in an amount effectiveto treat or reduce pruritis in the mammal.